Introduction and objectives: Colorectal cancer (CRC) is the second most frequent cancer in women and the third in men. Identification of the mechanisms of progression in these early CRC stages is ... [more ▼]

Introduction and objectives: Colorectal cancer (CRC) is the second most frequent cancer in women and the third in men. Identification of the mechanisms of progression in these early CRC stages is important to develop new diagnostic and therapeutic tools. Formalin-Fixed Paraffin-Embedded (FFPE) specimens are materials that enable proteomic clinical research. Hence our aim was to address the comparison of FFPE samples from early CRC stages patients using shotgun proteomic analysis. Methods: We performed a retrospective study on 36 CRC tissue samples (pT1N0M0, n=16 and pT2N0M0, n=20) compared together and with 40 control tissue samples (20 patients with diverticulitis, using paired inflamed (DI) and healthy tissue (DH)). Each tissue slice was macrodissected to enrich in epithelial cells. We used FFPE-FASP kit (Expedeon) for sample preparation and protein digests were analyzed using 2D-nanoAquity UPLC separation online with Q-Tof Synapt HDMSTM G2 using ion mobility as additional separation. We performed protein identification and differential analysis using Progenesis QI for proteomics (Nonlinear Dynamics). Results and discussion: We selected 149 proteins differentially distributed between T1 and T2 CRC stages which were not significantly different between CRC and DH or DI. Only 30 proteins were significantly more abundant in T1 versus T2 and 119 were distributed inversely, with a minimum fold ratio of 2. Among those, ATP synthase subunit beta, Aspartate-tRNA ligase, Haptoglobin and Kininogen were identified. . Moreover, we validated Kininogen and 3 others proteins with a significant differential distribution between pT1N0M0 and pT2N0M0 stages by immunohistochemistry. Conclusion: This FFPE retrospective study comparing T1 and T2 CRC highlighted proteins already previously identified as potential CRC biomarkers. These proteins may reflect important early changes in cancer development and may help understanding early tumor progression. [less ▲]

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated ... [more ▼]

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated with the risk of relapse and tissue damage progression. Endoscopy is costly and invasive. Hence biomarkers correlating with intestinal healing could improve disease management and potentially decrease the number of endoscopy when patients are in clinical remission. Aim: We aimed to identify potential biomarkers associated to CD mucosal healing and relapse after IFX withdrawals by a shotgun label-free proteomic study. Methods: We used the STORI1 clinical trial cohort (n=103) aiming at identifying markers associated to relapse prediction after Infliximab treatment withdrawals. We used serum samples of patients in clinical remission (at base line). We grouped these according to the degree of intestinal healing seen at endoscopy or according to relapse occurrence during the 28 month follow-up and composed pooled samples. We performed depletion of the 20 most abundant plasma proteins on each serum pools and ran a proteomic label-free differential analysis using 2D-nanoUPLC-MSE HDMS Synapt G2 for data acquisition. We performed different statistical analysis. Moreover, a Gene Ontology annotation was also performed for the potential biomarkers highlighted. Results and Discussion: We identified analysing these depleted serum pools 430 different proteins and 188 proteins common to all samples. Among these, 40 were found with a significant differential abundance in the groups compared. We selected some among the most significant one (ratio>1.3) or being by nature consistent with the context of this study (sample origin and clinical question addressed). For example, the C-reactive protein (CRP) was found with a significant Ratio of 2 between Relapsers and Non Relapsers. The other potential biomarkers associated to mucosal healing or to relapse prediction, were selected for further validation by Western Blot analysis (WB), routine laboratory tests and also by a Mass Spectrometry based technology: multiplexed selected reaction monitoring (SRM). Multiplexed SRM will enable quantitative analysis of these candidates in each individual patient as well as WB tests. Conclusions: This research strategy and the validation results on potential biomarkers associated to mucosal healing or relapse after treatment cessation in this cohort of CD patients, as well as tests done on other CD patients, might provide new opportunities for patient follow-up test developments. The next step is to perform SRM validation on the STORI cohort and design signatures using these potential biomarkers SRM data for prognosis power evaluation. 1. Louis E, Mary JY, Vernier-Massouille G, et al. Maintenance of remission among patients with Crohn's disease on antimetabolite therapy after infliximab therapy is stopped. Gastroenterology 2012;142:63-70 e5; quiz e31. [less ▲]

Genome-wide association studies of the related chronic inflammatory bowel diseases (IBD) known as Crohn's disease and ulcerative colitis have shown strong evidence of association to the major ... [more ▼]

Genome-wide association studies of the related chronic inflammatory bowel diseases (IBD) known as Crohn's disease and ulcerative colitis have shown strong evidence of association to the major histocompatibility complex (MHC). This region encodes a large number of immunological candidates, including the antigen-presenting classical human leukocyte antigen (HLA) molecules. Studies in IBD have indicated that multiple independent associations exist at HLA and non-HLA genes, but they have lacked the statistical power to define the architecture of association and causal alleles. To address this, we performed high-density SNP typing of the MHC in >32,000 individuals with IBD, implicating multiple HLA alleles, with a primary role for HLA-DRB1*01:03 in both Crohn's disease and ulcerative colitis. Noteworthy differences were observed between these diseases, including a predominant role for class II HLA variants and heterozygous advantage observed in ulcerative colitis, suggesting an important role of the adaptive immune response in the colonic environment in the pathogenesis of IBD. [less ▲]

BACKGROUND & AIMS: There is a need for a scoring system that provides a comprehensive assessment of structural bowel damage, including stricturing lesions, penetrating lesions, and surgical resection, for ... [more ▼]

BACKGROUND & AIMS: There is a need for a scoring system that provides a comprehensive assessment of structural bowel damage, including stricturing lesions, penetrating lesions, and surgical resection, for measuring disease progression. We developed the Lemann Index and assessed its ability to measure cumulative structural bowel damage in patients with Crohn's disease (CD). METHODS: We performed a prospective, multicenter, international, cross-sectional study of patients with CD evaluated at 24 centers in 15 countries. Inclusions were stratified based on CD location and duration. All patients underwent clinical examination and abdominal magnetic resonance imaging analyses. Upper endoscopy, colonoscopy, and pelvic magnetic resonance imaging analyses were performed according to suspected disease locations. The digestive tract was divided into 4 organs and subsequently into segments. For each segment, investigators collected information on previous operations, predefined strictures, and/or penetrating lesions of maximal severity (grades 1-3), and then provided damage evaluations ranging from 0.0 (no lesion) to 10.0 (complete resection). Overall level of organ damage was calculated from the average of segmental damage. Investigators provided a global damage evaluation (from 0.0 to 10.0) using calculated organ damage evaluations. Predicted organ indexes and Lemann Index were constructed using a multiple linear mixed model, showing the best fit with investigator organ and global damage evaluations, respectively. An internal cross-validation was performed using bootstrap methods. RESULTS: Data from 138 patients (24, 115, 92, and 59 with upper tract, small bowel, colon/rectum, and anus CD location, respectively) were analyzed. According to validation, the unbiased correlation coefficients between predicted indexes and investigator damage evaluations were 0.85, 0.98, 0.90, 0.82 for upper tract, small bowel, colon/rectum, anus, respectively, and 0.84 overall. CONCLUSIONS: In a cross-sectional study, we assessed the ability of the Lemann Index to measure cumulative structural bowel damage in patients with CD. Provided further successful validation and good sensitivity to change, the index should be used to evaluate progression of CD and efficacy of treatment. [less ▲]

AIM: The aim of this study is to determine the prevalence and evolution of anaemia in prospectively followed children and adolescents diagnosed with Crohn's disease (CD). METHODS: The BELCRO registry ... [more ▼]

AIM: The aim of this study is to determine the prevalence and evolution of anaemia in prospectively followed children and adolescents diagnosed with Crohn's disease (CD). METHODS: The BELCRO registry (inclusion May 2008-April 2010), describing current clinical treatment practice of children diagnosed with CD, provided data on age, height, body mass index (BMI), paediatric Crohn's disease activity index (PCDAI), therapy and haemoglobin (Hb) at diagnosis 12 and 24 months follow-up. Anaemia was defined as Hb < -2 sd, while severe anaemia was defined as Hb < -4 sd. Patients were classified as child </=13 and adolescent >13 years of age. RESULT: Ninety-six were included, 13 dropped out due to insufficient Hb data (37 females/46 males; median age 13.3 years, range 2.2-17.8 years). At diagnosis, the median Hb sd was -2.66 (-8.4; 1.07) and was correlated with the PCDAI (p = 0.013). At diagnosis, 51/83 (61 %) were anaemic and all had active disease. Hb z-score significantly improved (p < 0.0001) but 26/68 (38 %) remained anaemic at 12 months and 29/76 (38 %) at 24 months of follow-up. The correlation to the PCDAI disappeared. At 24 months, children were more likely to be anaemic. There was no difference in iron dose nor duration of iron supplements between children and adolescents. Iron treatment was more readily given to patients presenting with anaemia. Hb did not differ between patients with (n = 28) or without iron supplements. Half of the patients with persisting anaemia were given iron supplements, of which, only three were given intravenously. CONCLUSION: Anaemia remains an important extra-intestinal manifestation of CD in children. Physicians, lacking optimal treatment strategies, undertreat their patients. [less ▲]

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated ... [more ▼]

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated with the risk of relapse and tissue damage progression. Endoscopy is costly and invasive. Hence biomarkers correlating with intestinal healing could improve disease management. We aimed to identify potential biomarkers associated to CD mucosal healing by a shotgun proteomics label free study. Methods: We used the STORI clinical trial cohort (n=103) aiming at identifying markers associated to relapse prediction after Infliximab treatment withdrawals. We used serum samples of patients in clinical remission, grouped according to the degree of intestinal healing seen at endoscopy. We performed depletion of the 20 most abundant plasma proteins on each serum pools and ran a proteomics label free differential analysis using 2D-nanoUPLC-MSE HDMS Synapt G1 for data acquisition and Protein Lynks Global Server vs 2.4 for data analysis (Waters, Corp., Milford, USA). Results and Discussion: We obtained potential biomarkers and designed a multiplexed -selected reaction monitoring (SRM) method for validation of these candidates in each individual patient. The method may also be tested in an independent set of IBD patients with and without mucosal healing. Conclusions: This research strategy and results of SRM validation of potential biomarkers associated to mucosal healing in this cohort of CD patients as well as the tests done on other CD patients, may provide new opportunities for CD follow-up tests development. [less ▲]

Abstract The association of primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH) is known as an overlap syndrome (OS). OS can also be described in the setting of concomitant presence of AIH ... [more ▼]

Abstract The association of primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH) is known as an overlap syndrome (OS). OS can also be described in the setting of concomitant presence of AIH and PSC. These diseases can in some cases be associated with ulcerative colitis. In this case report we describe, to our knowledge, the first case in the literature of a young Caucasian male suffering from ulcerative colitis and an overlap syndrome consisting of an association between PSC-AIH, with the concomitant presence of a membranous glomerulonephritis. [less ▲]

OBJECTIVE: Glucocorticoids are powerful anti-inflammatory compounds that also induce the expression of leptin and leptin receptor (Ob-R) in synovial fibroblasts through TGF-betasignalling and Smad1/5 ... [more ▼]

OBJECTIVE: Glucocorticoids are powerful anti-inflammatory compounds that also induce the expression of leptin and leptin receptor (Ob-R) in synovial fibroblasts through TGF-betasignalling and Smad1/5 phosphorylation. Compound A (CpdA), a selective glucocorticoid receptor agonist, reduces inflammation in murine arthritis models and does not induce diabetes or osteoporosis, thus offering an improved risk:benefit ratio in comparison with glucocorticoids. Due to the detrimental role of leptin in OA pathogenesis, we sought to determine whether CpdA also induced leptin and Ob-R protein expression as observed with prednisolone. METHODS: Human synovial fibroblasts and chondrocytes were isolated from the synovium and cartilage of OA patients after joint surgery. The cells were treated with prednisolone, TGF-beta1, TNF-alpha and/or CpdA. Levels of leptin, IL-6, IL-8, MMP-1 and MMP-3 were measured by ELISA and expression levels of Ob-R phospho-Smad1/5, phospho-Smad2, alpha-tubulin and glyceraldehyde 3-phosphate dehydrogenase were analysed by western blotting. RESULTS: CpdA, unlike prednisolone, did not induce leptin secretion or Ob-R protein expression in OA synovial fibroblasts. Moreover, CpdA decreased endogenous Ob-R expression and down-regulated prednisolone-induced leptin secretion and Ob-R expression. Mechanistically, CpdA, unlike prednisolone, did not induce Smad1/5 phosphorylation. CpdA, similarly to prednisolone, down-regulated endogenous and TNF-alpha-induced IL-6, IL-8, MMP-1 and MMP-3 protein secretion. The dissociative effect of CpdA was confirmed using chondrocytes with no induction of leptin secretion, but with a significant decrease in IL-6, IL-8, MMP-1 and MMP-3 protein secretion. CONCLUSION: CpdA, unlike prednisolone, did not induce leptin or Ob-R in human OA synovial fibroblasts, thereby demonstrating an improved risk:benefit ratio. [less ▲]

Chronic diarrhoea is a frequent clinical presentation in our population. It may correspond to many gastrointestinal or systemic pathologies. Most frequent causes are irritable bowel syndrome, functional ... [more ▼]

Chronic diarrhoea is a frequent clinical presentation in our population. It may correspond to many gastrointestinal or systemic pathologies. Most frequent causes are irritable bowel syndrome, functional intestinal disorders or lactose intolerance, but organic diseases have also to be searched. Focused patient questioning and some specific aspects of clinical examination play a key-role in diagnosis orientation and the use of complementary explorations. The present paper proposes a structured diagnostic procedure aiming at an optimal use of complementary explorations. [less ▲]

BACKGROUND: While therapeutic strategies able to change the natural history of inflammatory bowel diseases (IBD) are being developed, factors predicting aggressive disease are needed to be able to choose ... [more ▼]

BACKGROUND: While therapeutic strategies able to change the natural history of inflammatory bowel diseases (IBD) are being developed, factors predicting aggressive disease are needed to be able to choose the appropriate therapeutic strategy for the individual patient based on the risk/benefit ratio. The aim of this review is to focus on the tools assisting the clinician in routine practice regarding the prediction of disease evolution. METHODS: A literature review was performed, which was mainly based on PubMed search, using the following terms: Crohn's disease, ulcerative colitis, inflammatory bowel disease, genetics, serology, biomarkers, endoscopy, C-reactive protein, faecal calprotectin, disease evolution and complications. RESULTS: For the prediction of disease evolution, clinical characteristics, particularly disease location and behaviour, are probably currently the most useful. In addition, a series of biomarkers, including genetic, serological and inflammatory markers, as well as characteristics of endoscopic lesions may have an added value. CONCLUSIONS: Simple clinical, biological and endoscopic tools may help the clinician in predicting disease evolution in IBD. However, these tools are still insufficient, and prospective evaluation of new genetic and biological markers are needed. [less ▲]

BACKGROUND AND OBJECTIVES: Infliximab is a monoclonal anti-tumor necrosis factor-alpha (anti-TNFalpha) antibody that profoundly modified the treatment of Crohn's disease (CD). The polymorphism of Fc fragment of IgG, low affinity IIIa, receptor (CD16a) [FCGR3A] influences the biological response to infliximab in patients with CD. Our aim was to study its influence on infliximab pharmacokinetics and risk of relapse after infliximab discontinuation. METHODS: In 111 CD patients in remission, infliximab was discontinued and its concentrations were measured for 30 months or until relapse. Infliximab pharmacokinetics were described using monocompartmental population modeling. RESULTS: The elimination rate of infliximab increased with C-reactive protein (CRP) [p = 0.00018] and was 16 % higher in FCGR3A-158V/V patients than in F carriers (p = 0.0028). Risk of relapse was higher in patients with baseline CRP >/=5 mg/L than in those with a lower value (p = 0.0000029). In addition, there was a first-order interaction between CRP and the FCGR3A genotype; in patients with high CRP, risk of relapse was higher for V/V patients than for F carriers (hazard ratio 4.80 and 2.84 for V/V and F carriers, respectively; p = 0.013). CONCLUSION: Both increased inflammation and FCGR3A-158V/V genotype are associated with increased infliximab elimination and risk of relapse after infliximab discontinuation in patients with CD. [less ▲]

INTRODUCTION: Our goals were to assess the prevalence of biological and tissue remission in routine practice in Crohn's disease, and to evaluate the correlation between biological or tissue remission and ... [more ▼]

INTRODUCTION: Our goals were to assess the prevalence of biological and tissue remission in routine practice in Crohn's disease, and to evaluate the correlation between biological or tissue remission and clinical or demographic characteristics as well as their impact on disease outcome. METHODS: We performed a retrospective monocenter study. Biological remission was defined by a CRP < 5 mg/I. Tissue remission was defined by the absence of ulcer at endoscopy and/or absence of signs of acute inflammation at MRI. Association with demographic, clinical and laboratory markers was studied by logistic regression models and rates of relapses, hospitalizations and surgeries were compared using the logrank test. RESULTS: Among the 263 patients included, 147 were in clinical remission; 102/147 (69%) were in biological remission. Fifty-six patients also had morphological evaluation: 37 (66%) were in tissue remission. Biological remission was associated with older age, higher hemoglobin and lower BMI. Tissue remission was associated with older age, lower platelets count, absence of previous surgery, and the use of immunosuppressant. Time-to-relapse was significantly longer in patients with biological remission and in patients with tissue remission as compared to patients without biological or tissue remission. CONCLUSIONS: Among the patients in clinical remission seen as outpatients, two thirds were either in biological and/or tissue remission. Biological and/or tissue remission was associated with a better outcome than clinical remission alone. [less ▲]

BACKGROUND: Crohn's disease (CD)-associated dysbiosis could predispose patients to relapse. Gut microbiota composition of patients from the prospective cohort study designed to identify predictive factors of clinical relapse after infliximab discontinuation (STORI Study) was investigated to determine the impact of dysbiosis in CD relapse. METHODS: Fecal samples from 33 patients with CD in this cohort were collected at baseline, 2 months, 6 months, and at the end of the follow-up period (19 relapsers and 14 nonrelapsers). Healthy volunteers subjects (n = 29) were used as a control group. The fecal microbiota composition was assessed using quantitative PCR, and comparisons between the patient groups were made at different time points using the Wilcoxon test. The analysis of the time-to-relapse was performed according to the baseline median level of each bacterial signal. RESULTS: Dysbiosis was observed in patients with CD compared with healthy subjects, and it was characterized by low mean counts of Firmicutes (Clostridium coccoides [P = 0.0003], C. leptum [P < 0.0001], and Faecalibacterium prausnitzii [P = 0.003]). Lower rates of Firmicutes were seen in relapsers compared with nonrelapsers. Moreover, a low rate of F. prausnitzii (P = 0.014) and a low rate of Bacteroides (P = 0.030) predicted relapse independently from high C reactive protein level (P = 0.0001). CONCLUSIONS: In this work, we report that CD-associated dysbiosis, characterized by a decrease in Firmicutes, correlates with the time-to-relapse after infliximab withdrawal. A deficit in some bacterial groups or species, such as F. prausnitzii, may represent a predictive factor for relapse. Restoring normobiosis in CD could be a new goal for optimal CD management. [less ▲]

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic ... [more ▼]

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic biomarkers. However, the critical step in this field is the ability to obtain an efficient and repeatable extraction using the limited quantities of material available for research in hospital biobanks. This work describes the evaluation of the peptide/protein extraction using FFPE sections treated by the following two methods before shotgun proteomic analysis: a commercial solution (FFPE-FASP) (filter aided sample preparation) and an antigen retrieval-derived protocol (On Slice AR). Their efficiencies and repeatabilities are compared using data-independent differential quantitative label-free analysis. FFPE-FASP was shown to be globally better both qualitatively and quantitatively than On Slice AR. FFPE-FASP was tested on several samples, and differential analysis was used to compare the tissues of diverticulitis patients (healthy and inflammatory tissues). In this differential proteomic analysis using retrospective clinical FFPE material, FFPE-FASP was reproducible and provided a high number of confident protein identifications, highlighting potential protein biomarkers. BIOLOGICAL SIGNIFICANCE: In clinical proteomics, FFPE is an important resource for retrospective analysis and for the discovery of biomarkers. The challenge for FFPE shotgun proteomic analysis is preparation by an efficient and reproducible protocol, which includes protein extraction and digestion. In this study, we analyzed two different methods and evaluated their repeatabilities and efficiencies. We illustrated the reproducibility of the most efficient method, FFPE-FASP, by a pilot study on diverticulitis tissue and on FFPE samples amount accessible in hospital biobanks. These data showed that FFPE is suitable for use in clinical proteomics, especially when the FFPE-FASP method is combined with label-free shotgun proteomics as described in the workflow presented in this work. [less ▲]